CAR-T cell targeting ALPPL2 and HER2 proteins and application thereof

Abstract

The invention discloses a chimeric antigen receptor targeting ALPPL2 and HER2 proteins, T lymphocytes modified by the chimeric antigen receptor and application of the chimeric antigen receptor and the T lymphocytes in preparation of products for killing or assisting in killing tumor cells. The receptor comprises a first antibody and a second antibody, wherein the first antibody is formed by connecting an ALPPL2 single-chain antibody and an SNIPRs signal molecule, and the second antibody is formed by connecting an HER2 single-chain antibody and an IL10 cell factor. According to the CAR-T cell disclosed by the invention, the off-target activity faced by conventional CAR-T is effectively reduced, the possibility of occurrence of cytokine storm is reduced, and the safety is higher; meanwhile, the autosecreted IL10 cell factor promotes CAR-T cell proliferation and effect functions, and the activity and durability of the CAR-T in killing tumor cells are obviously improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a chimeric antigen receptor targeting ALPPL2 and HER2 proteins, a modified T lymphocyte thereof, and uses of the chimeric antigen receptor and the HER2 chimeric antigen receptor. Background Art

[0002] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) is a newly developed cutting-edge cell therapy technology that has achieved unprecedented success in the treatment of hematological tumors, but the effect of CAR-T therapy in the treatment of solid tumors is not satisfactory. It is mainly manifested in: 1. Most solid tumor-related proteins are also expressed in normal tissues, and there are almost no solid tumor-specific surface antigens, which can easily lead to non-specific targeting or off-target activity of CAR-T cells, which may cause great harm or even death risks to patients. For example, in a clinical study of CAR-T cells targeting human epidermal growth factor receptor 2 (HER2), Morgan et al. found that CAR-T cells caused great toxicity to the patient's lungs half an hour after administration, causing the patient to die within a few days. 2. The persistence of anti-tumor response is highly correlated with the persistence of CAR-T cells, and antigen-independent CAR tonic signaling often causes conventional CAR-T cells to enter a terminal exhaustion state prematurely, resulting in the inability of CAR-T cells to survive in the tumor for a long time and exert tumor killing activity. 3. Solid tumors usually have an immunosuppressive tumor microenvironment. Once CAR-T cells reach the tumor and recognize antigens, the immunosuppressive tumor microenvironment may inhibit the activity of CAR-T cells, resulting in poor tumor killing effect.

[0003] Placental alkaline phosphatase-like protein 2 (ALPPL2) is hardly expressed in normal tissues, and is only expressed at a low level in the placenta, reproductive tissues, and lung tissues. Studies have shown that ALPPL2 expression increases in a variety of solid tumors, such as ovarian cancer, testicular cancer, endometrial cancer, non-small cell lung cancer, bladder cancer, gastric cancer, etc., and is related to cancer progression. It is a target with good tumor specificity and has the potential to be developed in the form of drugs such as ADC, bispecific antibodies, and CAR-T.

[0004] The human epidermal growth factor receptor 2 (HER2) gene is located on chromosome 17q21 and is a proto-oncogene. HER2 is lowly expressed or not expressed in normal adult tissues, but is amplified or overexpressed in multiple tumors such as breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, and prostate cancer. In addition, clinically, HER2 expression is significantly negatively correlated with the sensitivity of tumors to chemotherapy and biological therapy. These two characteristics make HER2 an ideal specific tumor treatment target and monitoring / prognosis marker.

[0005] At present, CAR-T in tumor treatment, especially solid tumor treatment, faces complex situations such as off-target activity, CAR-T cell exhaustion, and tumor suppressive microenvironment, which lead to poor clinical treatment effects and even patient death. How to overcome the above technical defects and better play the role of CAR-T cells in fighting solid tumors needs to be solved urgently. Summary of the invention

[0006] To overcome the above technical defects, the present invention discloses a chimeric antigen receptor targeting ALPPL2 and HER2 proteins, the receptor comprising a first antibody composed of an ALPPL2 single-chain antibody and a SNIPRs signaling molecule connected together, and a second antibody composed of a HER2 single-chain antibody and an IL10 cytokine connected together; the coding sequence of the ALPPL2 antibody is SEQ ID NO:1, and the amino acid sequence is SEQ ID NO:2; the coding sequence of the SNIPRs signaling molecule is SEQ ID NO:3, and the amino acid sequence is SEQID NO:4; the coding sequence of the HER2 single-chain antibody is SEQ ID NO:7, and the amino acid sequence is SEQ ID NO:8; the coding sequence of the IL10 cytokine is SEQ ID NO:9, and the amino acid sequence is SEQ ID NO:10.

[0007] As a preferred embodiment, the HER2 single-chain antibody and the IL10 cytokine sequence are connected by T2A, the T2A coding sequence is SEQ ID NO:11, and the amino acid sequence is SEQ ID NO:12.

[0008] The front end of the HER2 single-chain antibody of the present invention constructs a UAS structure, and together with miniCMV, constitutes a UAS-miniCMV promoter region, and its coding sequence is SEQ ID NO:13.

[0009] The other end of the SNIPRs signal molecule of the present invention connected to the ALPPL2 single-chain antibody is connected to the VP64 transcription factor, whose coding sequence is SEQ ID NO:5 and amino acid sequence is SEQ ID NO:6.

[0010] The present invention further discloses the use of the chimeric antigen receptor and the modified T lymphocytes thereof in preparing a product for killing or assisting in killing tumor cells, wherein the tumor cells are breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer or prostate cancer tumor cells.

[0011] Compared with the prior art, the present invention combines SNIPRs AND logic gates and IL10 autocrine strategies to successfully construct chimeric antigen receptors targeting ALPPL2 and HER2 proteins and their modified T lymphocytes. The CAR-T cell uses ALPPL2 as the first tumor recognition antigen and HER2 as the second tumor recognition antigen. Only when the tumor cell expresses both ALPPL2 and HER2 antigens, CAR-T activates the tumor killing function. Experiments have shown that when the CAR-T cell is activated, the expression of HER2-CAR stops 6-8 hours after it is out of contact with the first tumor antigen ALPPL2. This can greatly limit the activation and killing of CAR-T to the tumor microenvironment and avoid off-target activity. The overexpression of IL10 allows CAR-T cells to retain the complete mitochondrial structure and function in the tumor microenvironment, and increases oxidative phosphorylation in a mitochondrial pyruvate carrier-dependent manner, promotes the proliferation and effector function of CAR-T cells, and induces stem cell-like memory responses, thereby providing lasting protection against tumor re-attacks.

[0012] In summary, the CAR-T cells of the present invention effectively reduce the off-target activity faced by conventional CAR-T, reduce the possibility of cytokine storm, and have higher safety; at the same time, the autocrine IL10 structure promotes the proliferation and effector function of CAR-T cells, and significantly improves the activity and persistence of CAR-T in killing tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the sequence structure of the chimeric antigen receptor and its CAR-T cell in this example.

[0014] Figure 2 This is a schematic diagram of the mechanism by which CAR-T cells kill tumor cells in this example.

[0015] Figure 3 In the in vitro tumor killing ability verification test of CAR-T cells in this example, IFNγ, TNF

[0016] α, IL10 secretion.

[0017] Figure 4 In the in vitro tumor killing ability verification test of CAR-T cells in this example, flow cytometry was used to detect each

[0018] Graphical representation of the expression of HER2-CAR and CD137 on group A T cells.

[0019] Figure 5 This is a graphical representation of the results of detecting tumor cell killing ratio using a dual luciferase reporter gene detection kit in the in vitro tumor killing ability verification test of CAR-T cells in this example.

[0020] Figure 6 HER2-CAR expression half-life curve of CAR-T cells in this example. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific embodiments.

[0022] The purpose of this example is to construct and screen chimeric antigen receptors targeting ALPPL2 and HER2 proteins, and to prepare CAR-T cells AntiALPPL2-SNIPRs-IL10-HER2-CAR-T modified therefrom. It is further demonstrated that the cells have higher tumor cell specificity and longer-lasting effective activity than ordinary CAR-T cells.

[0023] 1. Construction of AntiALPPL2-SNIPRs-IL10-HER2-CAR-T

[0024] The AntiALPPL2-SNIPRs-IL10-HER2-CAR-T of this embodiment is composed of AntiALPPL2

[0025] L2-SNIPRs and IL10-HER2-CAR are carried by two different lentiviruses, and the sequence structures are as follows Figure 1 As shown. After the two lentiviruses are co-transfected into CD3-activated T cells, AntiALPPL2 is expressed on T cells. When AntiALPPL2 recognizes the tumor surface antigen ALPPL2, it activates the SNIPRs signaling molecule, hydrolyzes and releases the downstream connected VP64 transcription factor, and the VP64 transcription factor enters the cell nucleus and binds to the UAS structure at the front end of the IL10-HER2-CAR sequence, thereby driving the expression of HER2-CAR and IL10. When HER2-CAR recognizes the tumor surface antigen HER2, it mediates T cell activation and secretes tumor killing factors such as IFNγ, TNFα, Perforin, and Granzyme B, ultimately exerting tumor killing activity. The specific mechanism is as follows Figure 2 shown.

[0026] The technical process of the construction is as follows:

[0027] ①AntiALPPL2-SNIPRs plasmid, IL10-HER2-CAR plasmid construction→②Plasmid amplification→③Plasmid extraction→④Lentivirus preparation→⑤Peripheral blood PBMC isolation and activation→⑥Lentivirus transfection of T cells→ AntiALPPL2-SNIPRs-IL10-HER2-CAR-T cell expansion → AntiALPPL2-SNIPRs

[0028] -IL10-HER2-CAR-T cell function detection.

[0029] The construction plan is as follows:

[0030] 1. Construction and amplification of AntiALPPL2-SNIPRs plasmid and IL10-HER2-CAR plasmid

[0031] like Figure 1 As shown, the detailed structure of AntiALPPL2-SNIPRs in this embodiment includes MSCV promoter sequence, AntiALPPL2 sequence, SNIPRs sequence and VP64 transcription factor sequence in sequence. The detailed structure of IL10-HER2-CAR includes UAS sequence, miniCMV promoter sequence, HER2-CAR sequence, T2A sequence and IL-10 sequence in sequence.

[0032] The Novozyme P505-d1 PCR kit was used to PCR amplify AntiALPPL2-SNIPRs, IL10-HER2-

[0033] Each part of the CAR gene fragment and shuttle plasmid framework.

[0034] The AntiALPPL2-SNIPRs and IL10-HER2-CAR genes were synthesized by Shanghai Jierui Biotechnology Co., Ltd. The AntiALPPL2 coding sequence is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2; the SNIPRs coding sequence is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4; the VP64 coding sequence is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6; the HER2-scFv coding sequence is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:8; the IL10 coding sequence is shown in SEQ ID NO:9, and the amino acid sequence is shown in SEQ ID NO:10; the HER2-CAR and IL10 sequences are connected by T2A, the T2A coding sequence is shown in SEQ ID NO:11, and the amino acid sequence is shown in SEQ ID NO:12. The front end of the IL10-HER2-CAR sequence is a UAS structure, which forms a UAS-miniCMV promoter region with miniCMV, and the coding sequence is shown in SEQ ID NO:13.

[0035] The synthesized gene sequence was amplified by PCR and constructed into the lentiviral shuttle plasmid by homologous recombination. After the constructed shuttle plasmid was transformed into the competent cell DH5α strain, it was spread on an agar culture plate containing ampicillin and placed in a 37°C incubator for 15 hours. After the monoclonal strain was formed, 5 monoclones of each strain were picked and inoculated into 2mL LB medium containing ampicillin and placed in a 37°C shaker for 5 hours. 1μL of suspension was taken from each monoclonal strain for bacterial liquid PCR amplification of the target fragment, and the monoclone with the target fragment was determined by DNA electrophoresis. 1mL of monoclonal bacterial liquid with the target fragment was taken and sent to Shenggong Bioengineering Co., Ltd. for sequencing confirmation of the target fragment, and the remaining 1mL was stored in a 4°C refrigerator. Select a correctly recombined AntiALPPL2-SNIPRs and IL10-HER2-CAR gene monoclonal strain, take 350μL and inoculate it into 35mL LB medium containing ampicillin, and place it in a 37°C shaker for 15 hours.

[0036] 2. Plasmid extraction

[0037] The plasmid extraction kit used was the Tiangen plasmid medium-quantity small-scale plasmid extraction kit, and the specific operation scheme was referred to the product manual. The obtained shuttle plasmid was quantified and stored in a -20°C refrigerator for future use.

[0038] 3. Lentivirus Preparation

[0039] One day before transfection, inoculate 1.3E7 293T cells in a T75 culture flask and culture for 1 day until the cell density is about 90%. 1 hour before transfection, replace the medium with 10mL DMEM+10%FBS+1% double antibody+1%GlutaMAX+25mM HEPES medium. Prepare the packaging plasmid, membrane plasmid and shuttle plasmid and transfection reagent according to the instructions of the Lipo3000 transfection reagent kit, and place them at room temperature for 15 minutes to allow the DNA and transfection reagent to fully combine to form a stable transfection complex. Add the prepared DNA-transfection reagent mixture to the culture flask, mix gently, and place it in a 37°C incubator. After 8 hours, remove the culture medium, wash once with saline or PBS, add 10mL of fresh DMEM+10%FBS+1%GlutaMAX+25mM HEPES medium, and place it in a 37°C incubator for culture. 48 hours after transfection, the culture supernatant was collected into a 15 mL centrifuge tube, centrifuged at 4000 g for 5 min, the virus supernatant was collected, filtered through a 0.45 μm filter membrane, divided into cryopreservation tubes and stored in a -80 ° C refrigerator.

[0040] 4. Peripheral blood PBMC isolation and activation

[0041] Mix the obtained peripheral blood with saline at a ratio of 1:1, take a 15mL centrifuge tube, add 5mL of room temperature Ficoll, and gently add 8~9mL of peripheral blood mixture. Centrifuge at 800g for 20min, transfer the buffy coat to a new 15mL centrifuge tube. Add saline to 15mL, shake the cells, centrifuge at 500g for 5min, and discard the supernatant. Add saline to 15mL, resuspend the cells, centrifuge at 200g for 5min, and discard the supernatant. Resuspend the cells with 5mL of AIM-V medium containing 5% AB serum + 1% GlutaMAX + 25mM HEPES + 300 IU / mL IL-2, add CD3 / 28 magnetic beads according to the cell number and the ratio of cells: magnetic beads = 1:3, and place in a 37℃ incubator for activation for 48hr.

[0042] 5. Lentiviral transfection of T cells

[0043] One day before T cell activation, 12-well plates were coated with 15ug / mL Retronectin and placed in a 4°C refrigerator to avoid light overnight. On the day of transfection, the coated wells were removed and blocked with 2.5% HSA for 30 minutes. 2mL AntiALPPL2-SNIPRs virus and 2mL IL10-HER2-CAR virus were added to each well, and centrifuged at 32°C, 2000g for 90 minutes. The magnetic beads were adsorbed on a magnetic stand, CD3+T cells were collected, the unbound cell suspension was removed, and the magnetic beads and cells were resuspended in 3mL AIM-V medium containing 5% AB serum + 1% GlutaMAX + 25mMHEPES + 300 IU / mL IL-2. The magnetic bead cell suspension was blown, and the magnetic beads were adsorbed on a magnetic stand again. The magnetic beads were removed, the cell suspension was counted, and the cell concentration was adjusted to 1E6 Cells / mL. Take out the centrifuged plate, add 1 mL of activated T cell suspension to each well, centrifuge at 32°C, 2000g for 10 min, and place in a 37°C incubator for 24 hours. If you need to increase the transfection rate, repeat the transfection once after 24 hours.

[0044] 6. AntiALPPL2-SNIPRs-IL10-HER2-CAR-T cell expansion and functional detection

[0045] The transfected CAR-T cells were collected, resuspended in AIM-V medium containing 5% AB serum + 10ng / mL IL-7 + 10ng / mL IL-15 + 10ng / mL IL-21 + 1% GlutaMAX + 25mM HEPES, and cultured in a 37°C incubator for 48 hours. Some cells were taken for functional experiments and flow cytometry.

[0046] 2. Verification of the in vitro tumor killing ability of AntiALPPL2-SNIPRs-IL10-HER2-CAR-T cells.

[0047] The SKBR3 cell line is naturally ALPPL2-negative and HER2-positive, labeled as SKBR3 ALPPL2- / HER2+ . Overexpression of ALPPL2 gene in natural SKBR3 cells resulted in SKBR3 ALPPL2+ / HER2+ The coding sequence of ALPPL2 is SEQ ID NO: 14, and the amino acid sequence is SEQ ID NO: 15. CRISPR-Cas9 was used to knock out the HER2 gene of natural SKBR3 cells to obtain HER2-negative cells, which were labeled as SKBR3. ALPPL2- / HER2- The three pairs of sgRNA sequences used are: sgRNA-1F sequence as shown in SEQ ID NO: 16, sgRNA-1R sequence as shown in SEQ ID NO: 17, sgRNA-2F sequence as shown in SEQ ID NO: 18, sgRNA-2R sequence as shown in SEQ ID NO: 19, sgRNA-3F sequence as shown in SEQ ID NO: 20, and sgRNA-3R sequence as shown in SEQ ID NO: 21. In SKBR3 ALPPL2- / HER2- Overexpression of ALPPL2 gene in cells resulted in SKBR3 ALPPL2+ / HER2- All four groups of cells carry the Luciferase gene, whose coding sequence is shown in SEQ ID NO: 22 and whose amino acid sequence is shown in SEQ ID NO: 23, so as to facilitate in vitro detection of tumor killing activity.

[0048] SKBR3 ALPPL2- / HER2- SKBR3 ALPPL2+ / HER2- SKBR3 ALPPL2- / HER2+ SKBR3 ALPPL2+ / HER2+ The four groups of cells were resuspended in DMEM medium containing 10% FBS, the concentration was adjusted to 2E5 Cells / mL, and inoculated into 96-well plates, 100μL per well, 2E4 Cells / well. After 24 hours, the DMEM medium was aspirated. The cell concentrations of the five groups of MOCK-T, HER2-CAR-T, IL10-HER2-CAR-T, AntiALPPL2-SNIPRs-HER2-CAR-T, and AntiALPPL2-SNIPRs-IL10-HER2-CAR-T were adjusted to 5E5 Cells / mL, and 200μL of T cells from each group were co-cultured with tumor cells, with a final effector-target ratio of 5:1. After 36 hours, the culture supernatant was collected, and the secretion of IFNγ, TNFα, and IL10 was detected by ELISA. The results are as follows Figure 3The expression of HER2-CAR and CD137 in each group of T cells was detected by flow cytometry. The results are shown in Figure 4 The dual luciferase reporter gene detection kit was used to detect the tumor cell killing ratio. The results are shown in Figure 5 shown.

[0049] The results showed that HER2-CAR-T and IL10-HER2-CAR-T expressed positive HER2-CAR in all co-culture wells; ALPPL2- / HER2+ SKBR3 ALPPL2+ / HER2+ Two groups of HER2 + In the co-culture system, IFNγ, TNFα, and CD137 were all positive, and the tumor killing ratio was between 84.5% and 98.6%. In addition, IL10-HER2-CAR-T secreted IL10 in all co-culture wells.

[0050] PRs-HER2-CAR-T, AntiALPPL2-SNIPRs-IL10-HER2-CAR-T in SKBR3 ALPPL2+ / HER2- SKBR3 ALPPL2+ / HER2+ Two groups of ALPPL2 + In the co-culture wells, HER2-CAR, IFNγ, TNFα, and CD137 were positively expressed, and the tumor killing ratio was between 53.0 and 96.5%, and AntiALPPL2-SNIPRs-IL10

[0051] -HER2-CAR-T group had a significantly better tumor killing rate than AntiALPPL2-SNIPRs-HER2-CAR-T group. ALPPL2- / HER2- SKBR3 ALPPL2- / HER2+ Two groups of ALPPL2 - In the co-culture wells, HER2-CAR, IFNγ, TNFα, and CD137 expression were negative. ALPPL2+ / HER2- SKBR3 ALPPL2+ / HER2+ Two groups of ALPPL2 + IL10 was secreted in all co-culture wells.

[0052] 3. Detect the half-life of HER2-CAR expression in AntiALPPL2-SNIPRs-IL10-HER2-CAR-T cells.

[0053] When designing the AntiALPPL2-SNIPRs sequence, the present invention integrates the Myc sequence into the front end of the ALPPL2-scFv structure to form AntiALPPL2-SNIPRs containing the Myc sequence. Therefore, after Myc binds to the α-Myc antibody, it can also activate the downstream signal of AntiALPPL2-SNIPRs, and ultimately control the expression of HER2-CAR. In this experiment, the α-Myc antibody was coated on a 24-well plate, with a total of 10 wells. After 24 hours, 1 mL of AntiALPPL2-SNIPRs-IL10-HER2-CAR-T cells were added, 1E6 Cells / mL. Every 2 hours, a well of cells was aspirated, 300g, centrifuged for 5 minutes, the supernatant was removed, and the cells were resuspended with a new AIM-V medium containing 5% AB serum + 1% GlutaMAX + 25mM HEPES, and transferred to a new well without coating for culture to get out of contact with the α-Myc antibody. The expression of HER2-CAR at different time points was detected by flow cytometry. The HER2-CAR expression rate of the group that did not separate from the culture was corrected to 100%, and the other groups were corrected in turn to obtain the HER2-CAR expression rate at different time points after separation from the α-Myc antibody. The time and percentage curves were fitted to obtain the half-life of HER2-CAR expression. The t1 / 2 was about 6-8 hours after separation from the α-Myc antibody. The results are shown in Figure 6 shown.

[0054] From the above test results, it can be seen that the AntiALPPL2-SNIPRs-IL10-HER2-CAR-T group has a significantly better tumor killing ratio than the AntiALPPL2-SNIPRs-HER2-CAR-T group. When the CAR-T cells of the present invention are activated, 6-8 hours after they are out of contact with the first tumor antigen ALPPL2, the expression of HER2-CAR stops. This can greatly limit the activation and killing of CAR-T to the tumor microenvironment and avoid off-target activity. The problems of off-target activity and cell exhaustion of existing CAR-T and the inability to deliver anti-tumor effects for a long time have been successfully solved.

Claims

1. A chimeric antigen receptor targeting ALPPL2 and HER2 proteins, characterized in that: The chimeric antigen receptor includes a first antibody composed of an ALPPL2 single-chain antibody and a SNIPRs signaling molecule, and a second antibody composed of a HER2 single-chain antibody and an IL10 cytokine; the coding sequence of the ALPPL2 antibody is SEQ ID NO:1, and the amino acid sequence is SEQ ID NO:2; the coding sequence of the SNIPRs signaling molecule is SEQ ID NO:3, and the amino acid sequence is SEQ ID NO:4; the coding sequence of the HER2 single-chain antibody is SEQ ID NO:7, and the amino acid sequence is SEQ ID NO:8; the coding sequence of the IL10 cytokine is SEQ ID NO:9, and the amino acid sequence is SEQ ID NO:

10.

2. The chimeric antigen receptor according to claim 1, wherein The HER2 single-chain antibody and the IL10 cytokine sequence are connected by T2A, the T2A coding sequence is SEQ ID NO: 11, and the amino acid sequence is SEQ ID NO:

12.

3. The chimeric antigen receptor according to claim 1, wherein The front end of the HER2 single-chain antibody constructs a UAS structure, and together with miniCMV, forms a UAS-miniCMV promoter region, and its coding sequence is SEQ ID NO:

13.

4. The chimeric antigen receptor according to claim 1, wherein The other end of the SNIPRs signal molecule connected to the ALPPL2 single-chain antibody is connected to the VP64 transcription factor, whose coding sequence is SEQ ID NO:5 and amino acid sequence is SEQ ID NO:

6.

5. A CAR-T cell targeting ALPPL2 and HER2 protein, characterized in that: The cell is a T lymphocyte modified by the chimeric antigen receptor according to claim 1, 2, 3 or 4.

6. Use of the chimeric antigen receptor according to any one of claims 1 to 4 in the preparation of a product for killing or assisting in killing tumor cells, wherein the tumor cells are breast cancer tumor cells.

7. Use of the CAR-T cells of claim 5 in preparing a product for killing or assisting in killing tumor cells, wherein the tumor cells are breast cancer tumor cells.

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